Project Summary/Abstract: We request support to replace a defunct six-year old cryoprobe on our Agilent 600 NMR system. The cryoprobe was an outstanding performer with unparallel S/N ratio. Despite careful maintenance and annual servicing, the probe could not be repaired as Agilent has terminated its NMR division in 2013, and their subsequent seven-year servicing commitment/ability has expired after six years. Thus, we ask for funds to purchase a new Bruker 600 MHz cryoprobe, which also needs a new console to operate it. The replacement cryoprobe should also have the capability to pulse 19F together with 1H, 13C and 15N. The requested new quadruple-resonance cryoprobe would not only make the existing shielded 600 MHz system working again but also will enable us to perform recently developed 19F-13C TROSY and 1H-19F NOESY experiments. It is now possible to express proteins with 19F/13C labeled aromatic side chains (Tyr, Phe, Trp), nucleic acids with 19F/13C labeled bases (Adenine, Uracil), and also equally labeled ATP and AMP-PNP. We have shown that the 19F-13C TROSY experiments yield very sharp signals for tyrosine side chains in large protein systems. Together with new 1H-19F NOESY experiments and methyl TROSY this technology will provide access to monitor the dynamic state of aromatic side chains, record aromatic-methyl contacts and follow their changes upon protein interactions with ligands or other proteins, such as in transmembrane signal transduction. As fluorine doesn't occur naturally in biological macromolecules experiments can be recorded essentially background free. The new technology is particularly interesting for studies of translation initiation complexes, which are established targets for anti-tumor agents and are actively research here. Elucidating interactions and dynamics of bulky side chains (aromatics, methyls) and their changes promises new insights into the mechanism of transmembrane signal transduction of G-protein coupled receptors (GPCRs), which represent a large class of drug targets. 19F-based experiments will also facilitate studies of viral proteins in their membrane bound states. Following aromatic side chains has been very difficult in large systems so far. The 19F-13C TROSY, 1H-19F NOE difference spectroscopy, and their combination open new opportunities to address structural and functional questions of small RNAs and protein/RNA complexes. In addition, 19F/13C labeled small nucleotides (ATP, GTP etc.) are predicted to have extremely sharp signals and should be visible in very large systems and useful for following biological processes at native solution conditions.